SUPPLEMENTARY INFORMATION doi:10.1038/nature19814 Figure 3e - - - Beads: Hep SA Sup Hep SA Sup Hep - SA - Sup 150 102 76 102 76 Blot: NP-1 Blot: MECA-32 Blot: VEGF Figure 3f Rbt IgG ctrl IP VEGF IP Extended Data Figure 9d Beads: CD4-Fc SA Hep SA Hep Loading PLVAP 250 150 50 Blot: MECA-32 + 1M NaCl 50 Rbt IgG ctrl IP VEGF IP VEGF (dimer; non-reducing gel) 250 150 50 Beads: CD4-Fc SA Hep SA Hep Loading Blot:anti-human IgG Blot: VEGF Figure 3g Beads: SA Hep CD4-Fc SA Hep Loading Extended Data Figure 9e Beads: Hep Hep Hep SA 150 50 Blot:anti-human IgG Competition - FN ColI - Blot:anti-human IgG Supplementary Figure 1. The gel source data. The uncropped original blots from electrophoretic experiments. Shown in Fig.3e, 3f, 3g, Extended Data Fig. 9d and 9e WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Table 1: Full flow cytometric gating strategy of progenitor and myeloid leukocytes Adults Tissue leukocyte type Full gating used (a,b,c,d,e) Additional markers used References Spleen Embryonic-derived tissue resident macrophages Live/CD45 neg /CD8 neg /CD4 CD206 + / CSF-1R Bone marrow-derived macrophages Live/CD45 neg /CD8 neg /CD4 /F4/80 Int CSF-1R + 7,11,20 Recently entered tissue monocytes Live/CD45 neg /CD8 neg /CD4 neg / F4/80 Int /CD11b /Ly6c Low or hi CSF-1R + 20,50 Peritoneal macrophages Embryonic-derived tissue resident macrophages Live/CD45 neg /CD8 neg /CD4 /F4/80 CSF-1R + MHC II 7,11,20,25 low/neg 11,20,51-54 Bone marrow -derived macrophages Lung Live/CD45 neg /CD8 neg /CD4 Int CSF-1R + MHC II + 11,20,51,52 Embryonic-derived alveolar macrophages Live/CD45 neg/ CD8 neg /CD4 + /CD11c F4/80 high /CD206 + /SiclecF high /CSF-1R +7,11,18,20 Blood Bone marrow-derived infiltrating monocytes Live/CD45 + /Ly6C 26,55 Bone marrow-derived patrolling monocytes Live/CD45 + /Ly6C Peripheral lymph node Low 26,55 Bone marrow-derived macrophages Live/CD45 + /CD11b Int Colon Bone marrow-derived macrophages Live/CD45 + /CD11b Int 23,56 Liver Embryonic-derived tissue resident macrophages Live/CD45 neg /CD8 neg /CD4 Bone marrow Low or hi /Ly6c CSF-1R + 7,11,20,57 Bone marrow-derived macrophages Live/CD45 neg /CD8 neg /CD4 /F4/80 Int CSF-1R + 7,11,20,57 Recently entered tissue monocytes Live/CD45 neg /CD8 neg /CD4 neg / F4/80 Int /CD11b 20 Hematopoietic stem cells (HSC) Live/Lin neg /C-kit + /Sca-1 + 58 Common myeloid progenitor (CMP) Live/Lin neg /C-kit + /Sca-1 Neg Neg /IL7R 58 Common lymphoid progenitor (CCP) Live/Lin neg /C-kit + /Sca-1 Low /IL7R + 58 Ly6C high monocytes Live/CD45 + /CD11b + /Ly6C 59 Ly6C low monocytes Live/CD45 + /CD11b + Low /Ly6C 59 Brain Embryonic-derived microglia cells Live/CD45 Low / F4/80 6,7,20,60 Mammary gland Macrophages Live/CD45 neg /CD4 + 2 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Embryos Tissue leukocyte type Full gating used Additional markers used References Spleen Yolk sac-derived embryonic macrophages Live/CD45 CSF-1R + 7,11,20,25 Fetal liver-derived embryonic macrophages Live/CD45 Int CSF-1R + 7,11,20,25 Lung Yolk sac-derived embryonic macrophages Live/CD45 CSF-1R + 7,11,20,61 Fetal liver-derived embryonic macrophages Live/CD45 Int CSF-1R + 7,11,20 Blood Blood monocytes Live/CD45 + /Ly6C 26,55 Yolk sac-derived embryonic macrophages Live/CD45 Ly6C neg /CSF-1R + 7,11,20 Fetal liver-derived embryonic macrophages Live/CD45 /F4/80 Int Ly6C + /CSF-1R + 7,11,20 Macrophage-dendritic cell progenitor (MDP) Live/CD45 + /CD11b neg /CSF-1R + /c-kit + /Flt-3 + Neg /Ly6C 17,62 Fetal liver myeloid precursor (MP) Live/CD45 + /CD11b neg /CSF-1R + /c-kit + /Flt-3 Neg Neg /Ly6C 17,62 Common monocyte precursor (cmop) Live/CD45 + /CD11b neg /CSF-1R + /c-kit + /Flt-3 Neg /Ly6C + 17,62 Fetal liver Ly6C + monocytes Live/CD45 + /CD11b + /CSF-1R + /c-kit neg /Flt-3 Neg /Ly6C + 17,62 Fetal liver Ly6C neg monocytes Live/CD45 + /CD11b + /CSF-1R + /c-kit neg /Flt-3 Neg Neg /Ly6C 17,62 Erythro-myeloid progenitor (EMP) Live/CD41 + /CD45 + /c-kit Neg /F4/80 10,17 Hematopoietic stem cells (HSC) Live/c-kit + /Sca-1 + 10,17,63 Brain Embryonic-derived microglia cells Live/CD45 Low / F4/80 6,7,16,20,60 Yolk sac Yolk sac embryonic macrophages Live/CD45 + /CD11b CSF-1R + 7,8,11,20 Yolk sac embryonic macrophages (MAC) Live/CD41 + /CD45 /c-kit Neg CSF-1R + 10,21 Erythro-myeloid progenitor (EMP) Live/CD41 + /CD45 + /c-kit /F4/80 Neg CSF-1R + 10,17 a) The selected markers by which the cell types are referred to in the text are shown in bold. b) Live = 7-Amino-Actinomycin (7-AAD) negative c) Int = intermediate, neg = negative, d) Lin = Linage marker (CD3e, CD11b, B220, TER-119, Ly6G/Ly6C; 558074, BD) e) Lymphocytes from different adult tissues were all gated as follows: B lymphocytes Live/CD45 + /CD8 neg /CD4 neg /B220 + CD4 T-helper lymphocytes Live/CD45 neg /CD8 neg /CD4 + CD8 T-cytotoxic lymphocytes Live/CD45 neg /CD4 neg /CD8 + 50. Swirski, F. K. et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325, 612 616 (2009). 51. Rosas, M. et al. The transcription factor Gata6 links tissue macrophage phenotype and proliferative renewal. Science 344, 645 648 (2014). 52. Nguyen, H. H., Tran, B. T., Muller, W. & Jack, R. S. IL-10 acts as a developmental switch guiding monocyte differentiation to macrophages during a murine peritoneal infection. J. Immunol. 189, 3112 3120 (2012). 53. Gautier, E. L. et al. Gata6 regulates aspartoacylase expression in resident peritoneal WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION 53. Gautier, E. L. et al. Gata6 regulates aspartoacylase expression in resident peritoneal macrophages and controls their survival. J. Exp. Med. 211, 1525 1531 (2014). 54. Ghosn, E. E. et al. Two physically, functionally, and developmentally distinct peritoneal macrophage subsets. Proc. Natl Acad. Sci. USA 107, 2568 2573 (2010). 55. Geissmann, F., Jung, S. & Littman, D. R. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity 19, 71 82 (2003). 56. Bain, C. C. et al. Resident and pro-inflammatory macrophages in the colon represent alternative context-dependent fates of the same Ly6Chi monocyte precursors. Mucosal Immunol. 6, 498 510 (2013). 57. Movita, D. et al. Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages. J. Leukoc. Biol. 92, 723 733 (2012). 58. Adolfsson, J. et al. Identification of Flt3+ lympho-myeloid stem cells lacking erythromegakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, 295 306 (2005). 59. Hanna, R. N. et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C- monocytes. Nat. Immunol. 12, 778 785 (2011). 60. Gomez Perdiguero, E., Schulz, C. & Geissmann, F. Development and homeostasis of resident myeloid cells: the case of the microglia. Glia 61, 112 120 (2013). 61. Zaynagetdinov, R. et al. Identification of myeloid cell subsets in murine lungs using flow cytometry. Am. J. Respir. Cell Mol. Biol. 49, 180 189 (2013). 62. Hettinger, J. et al. Origin of monocytes and macrophages in a committed progenitor. Nat. Immunol. 14, 821 830 (2013). 63. Mebius, R. E. et al. The fetal liver counterpart of adult common lymphoid progenitors gives rise to all lymphoid lineages, CD45+CD4+CD3 cells, as well as macrophages. J. Immunol. 166, 6593 6601 (2001). 4 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 2: Antibodies used in flow cytometry and immunohistochemistry Flow cytometry Antigen Antibody clone Fluorochrome a) Catalog # Company b) B220 RA3-6B2 PE 553089 BD B220 RA3-6B2 BV421 562922 BD B220 RA3-6B2 Pacific Blue 558108 BD CD4 GK1.5 APC-Cy7 552051 BD CD4 RM4-5 A647 557681 BD CD4 RM4-5 PE 553048 BD CD8 53-6.7 PerCP-Cy5,5 551162 BD CD8 53-6.7 PE 553032 BD CD8 53-6.7 BV650 563234 BD CD11b M1/70 APC-Cy7 561039 BD CD11b M1/70 BB515 564454 BD CD11b M1/70 PE 553311 BD CD11c HL3 BV421 562782 BD CD11c HL3 PerCP-Cy5,5 560584 BD CD34 HM34 PerCP-Cy5,5 128607 Biolegend CD41 MWReg30 PE 561850 BD CD41 MWReg30 FITC 553848 BD CD45 30-F11 APC-Cy7 557659 BD CD45 30-F11 PerCP-Cy5,5 557659 BD CD45 30-F11 V500 561487 BD CD115 (CSF-1R) AFS98 Pe-Cy7 25-1152-80 ebiosciences CD117 (C-kit) ACK2 BV510 135119 Biolegend CD127 (IL-7) SB/199 Pe-Cy7 560733 BD CD135 (Flt-3) A2F10.1 BV421 562898 BD CD206 MR5D3 A488 MCA2235A488 Abd Serotec CD304 3DS304M PE 12-3041-82 ebiosciences F4/80 Cl:A3-1 A647 MCA497A647 Abd Serotec F4/80 Cl:A3-1 PE Ab105156 Abcam Lin 145-2C11, M1/70, RA3-6B2, APC 558074 BD TER-119, RB3-8C5 Ly6C AL-21 BV421 562727 BD Ly6C AL-21 FITC 553104 BD Ly6G 1A8 BV510 127633 Biolegend MHCII M5/114.15.2 Pe-Cy7 25-5321-82 ebiosciences Sca-1 177228 PE FAB1226P R&D Systems Siglec F E50-2440 PE-CF594 5627 BD VEGFR1 141522 PE FAB4711P R&D Systems VEGFR2 Avas12a1 PE 12-5821-81 ebiosciences Isotype controls Armenian Hamster IgG1, λ1 PerCP-Cy5,5 560554 BD Armenian Hamster IgG1, λ2 BV421 562919 BD Armenian Hamster IgG PerCP-Cy5,5 400931 Biolegend WWW.NATURE.COM/NATURE 5
RESEARCH SUPPLEMENTARY INFORMATION Rat IgG1 FITC 553924 BD Rat IgG2a A488 MCA1212A488 Abd Serotec Rat IgG2a A647 557690 BD Rat IgG2a BV421 562602 BD Rat IgG2a BV510 562952 BD Rat IgG2a BV650 56326 BD Rat IgG2a Pacific Blue 558109 BD Rat IgG2a PE-CF594 562302 BD Rat IgG2a PerCP-Cy5,5 550765 BD Rat IgG2a PE-Cy7 25-4321-81 ebiosciences Rat IgG2b APC-Cy7 552773 BD Rat IgG2b A647 400626 Biolegend Rat IgG2b BB515 564421 BD Rat IgG2b PerCP-Cy5,5 550764 BD Rat IgG2b V500 560784 BD Rat IgG2b BV510 135119 Biolegend Rat IgG2b V500 560784 BD Rat IgG2b BV510 135119 Biolegend Rat IgG2b PE-Cy7 25-4031-81 ebiosciences Rat IgM FITC 553942 BD Lineage Isotype Control Cocktail APC 558074 BD Immunohistochemistry Antigen Antibody clone Fluorochrome Catalog # Source Caveolin Rabbit polyclonal unconjucated sc-894 Santa-Cruz CD31 MEC13.3 APC 102510 BioLegend CD117 2B8, Rat IgG2b unconjucated 553352 BD F4/80 BM8 A488 MF48020 Invitrogen Lyve-1 Rabbit polyclonal unconjucated 102-PA50AG/103- Reliatech PA50 MAdCAM-1 MECA-367, Rat IgG2a unconjucated E.Butcher Stanford Univ. Plvap Meca-32, Rat IgG2a unconjucated MECA32-17-8 AK Invivo 2028/01 Plvap Meca-32, Rat IgG2a unconjucated 550563 BD Plvap PAL-E unconjucated ab8086 Abcam VEGF Rabbit polyclonal unconjucated Ab46154 Abcam Second stage antibodies Donkey anti Rat IgG A488 A21208 Life Technologies Goat anti Mouse IgG A488 A11029 Invitrogen Goat anti Rabbit IgG A546 A11035 Invitrogen Goat anti Rat IgG A546 A11081 Life Technologies Goat anti Rat IgG A647 A21247 Life Technologies Goat anti Rabbit IgG A633 A21071 Invitrogen a) APC, Allophycocyanin; APC-Cy7, Allophycocyanin-indotricarbocyanine; A488, Alexa Flour 488; A647, Alexa Fluor 647; BB515, Brilliant Blue 515; BV421,Brilliant Violet 421; BV510, Brilliant Violet 510; BV650, Brilliant Violet 650; FITC, Fluorescein isothiocyanate; PE, Phycoerythrin; Pe-Cy7,Phycoerythrin-indotricarbocyanine; PerCP-Cy5,5, Peridinin chlorophyll protein cyanine 5.5 b) BD, Becton-Dickinson 6 WWW.NATURE.COM/NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Table 3: Cell numbers in WT and Plvap / mice (a) Embryos WT Plvap / E10.5 Yolk-sac Total leukocytes (x10 6 ) 0.16 ± 0.02 0.13 ± 0.019 E12.5 Liver Total leukocytes (x10 6 ) 2.84 ± 0.48 2.76 ± 1.54 HSC 3024 ± 685.6 1951 ± 357.3 Yolk-sac Total leukocytes (x10 6 ) 2.81 ± 0.29 2.82 ± 0.665 E14.5 Liver Total leukocytes (x10 6 ) 21.39 ± 1.76 20.80 ± 2.30 CD11b F4/80 Int 3388 ± 373.0 6848 ± 11 b CD11b + F4/80 5280 ± 577.4 4563 ± 1419 Lung Total leukocytes (x10 6 ) 1.22 ± 0.47 0.88 ± 0.19 b E16.5 Liver Total leukocytes (x10 6 ) 39.43 ± 7.46 36.73 ± 9.83 CD11b + F4/80 Int 3970 ± 235.4 6421.5 ± 492.7 CD11b F4/80 3728 ± 210.5 3620 ± 290.2 Blood CD11b + Ly6C + 448.7 ± 164.0 185.3 ± 10.31 b Lung CD11b + F4/80 Int 5838 ± 214.3 3941 ± 440.3 CD11b F4/80 2406 ± 171.6 4149 ± 549.3 Spleen CD11b + F4/80 Int 8339 ± 1065 2824 ± 716.8 b CD11b F4/80 9046 ± 1607 5668 ± 1385 Adults Blood (Cells x 10 3 / ml of blood) WT Plvap / White blood cells 8.33 ± 1.04 8.70 ± 1.80 Lymphocytes 5.57 ± 0.54 6.87 ± 1.23 Monocytes 0.17 ± 0.04 0.22 ± 0.11 Neutrophils 2.56 ± 0.60 1.62 ± 0.60 WWW.NATURE.COM/NATURE 7
RESEARCH SUPPLEMENTARY INFORMATION Spleen Weight (g) 0.133 ± 0.028 0.083 ± 0.024 Total leukocytes after red blood lysis (x10 6 ) 42.35 ± 10.51 33.74 ± 13.89 CD11b F4/80 Int 2972 ± 441.3 3162 ± 449.3 CD11b + F4/80 2623 ± 351.7 653.3 ± 69.71 b B cells 24740 ± 5663 29482 ± 3299 Liver Weight (g) 1.47 ± 0.156 1.36 ± 0.27 CD11b F4/80 Int 1783 ± 420.6 3803 ± 812.0 CD11b + F4/80 2882 ± 486.8 546.0 ± 216.0 b B cells 4435 ± 220.8 4421 ± 842.6 Lung CD11b + CD11c 1163 ± 169.7 335.2 ± 35.57 b Peritoneal cells CD11b F4/80 1.6 X 10 6 ± 33553 349.8 ± 142.3 b Brain CD45 low F4/80 HI 37721 ± 5998 41132 ± 6906 PLN Total leukocytes (x10 6 ) 4.78 ± 1.78 5.76 ± 1.26 B cells 8319 ± 410.2 13864 ± 1537 b Bone marrow Total leukocytes (x10 6 ) 15.37 ± 1.91 12.83 ± 1.82 Mammary gland CD11b + F4/80 + 13649 ± 2687 3319 ± 281.4 b a) Cell numbers are shown as mean±sem (n=3-5/mice/genotype), determined from equal-sized tissue pieces or equal volumes of blood (the values can be compared between the two genotypes on each row, but not between the different rows). not between the diff b) p<0,05 8 WWW.NATURE.COM/NATURE